Fang-Ming Zhao

CHIMERIC FLORAL ORGANS1, Encoding a Monocot-Specific MADS Box Protein, Regulates Floral Organ Identity in Rice

The control of floral organ identity by homeotic MADS box genes is well established in eudicots. However, grasses have highly specialized outer floral organs, and the identities of the genes that regulate the highly specialized outer floral organs of grasses remain unclear. In this study, we characterized a MIKC-type MADS box gene, CHIMERIC FLORAL ORGANS (CFO1), which plays a key role in the regulation of floral organ identity in rice (Oryza sativa). The cfo1 mutant displayed defective marginal regions of the palea, chimeric floral organs, and ectopic floral organs. Map-based cloning demonstrated that CFO1 encoded the OsMADS32 protein. Phylogenetic analysis revealed that CFO1/OsMADS32 belonged to a monocot-specific clade in the MIKC-type MADS box gene family. The expression domains of CFO1 were mainly restricted to the marginal region of the palea and inner floral organs. The floral organ identity gene DROOPING LEAF (DL) was expressed ectopically in all defective organs of cfo1 flowers. Double mutant analysis revealed that loss of DL function mitigated some of the defects of floral organs in cfo1 flowers. We propose that the CFO1 gene plays a pivotal role in maintaining floral organ identity through negative regulation of DL expression.

Rolling-leaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves

As an important agronomic trait, leaf rolling in rice (Oryza sativa L.) has attracted much attention from plant biologists and breeders. Moderate leaf rolling increases the amount of photosynthesis in cultivars and hence raises grain yield. Here, we describe the map-based cloning of the gene RL14, which was found to encode a 2OG-Fe (II) oxygenase of unknown function. rl14 mutant plants had incurved leaves because of the shrinkage of bulliform cells on the adaxial side. In addition, rl14 mutant plants displayed smaller stomatal complexes and decreased transpiration rates, as compared with the wild type. Defective development could be rescued functionally by the expression of wild-type RL14. RL14 was transcribed in sclerenchymatous cells in leaves that remained wrapped inside the sheath. In mature leaves, RL14 accumulated mainly in the mesophyll cells that surround the vasculature. Expression of genes related to secondary cell wall formation was affected in rl14-1 mutants, and cellulose and lignin content were altered in rl14-1 leaves. These results reveal that the RL14 gene affects water transport in leaves by affecting the composition of the secondary cell wall. This change in water transport results in water deficiency, which is the major reason for the abnormal shape of the bulliform cells.

MULTI-FLORET SPIKELET1 , Which Encodes an AP2/ERF Protein, Determines Spikelet Meristem Fate and Sterile Lemma Identity in Rice

MULTI-FLORET SPIKELET1 determines spikelet meristem fate and sterile lemma identity in rice. The spikelet is a unique inflorescence structure of grass. The molecular mechanism that controls the development of the spikelet remains unclear. In this study, we identified a rice (Oryza sativa) spikelet mutant, multi-floret spikelet1 (mfs1), that showed delayed transformation of spikelet meristems to floral meristems, which resulted in an extra hull-like organ and an elongated rachilla. In addition, the sterile lemma was homeotically converted to the rudimentary glume and the body of the palea was degenerated in mfs1. These results suggest that the MULTI-FLORET SPIKELET1 (MFS1) gene plays an important role in the regulation of spikelet meristem determinacy and floral organ identity. MFS1 belongs to an unknown function clade in the APETALA2/ethylene-responsive factor (AP2/ERF) family. The MFS1-green fluorescent protein fusion protein is localized in the nucleus. MFS1 messenger RNA is expressed in various tissues, especially in the spikelet and floral meristems. Furthermore, our findings suggest that MFS1 positively regulates the expression of LONG STERILE LEMMA and the INDETERMINATE SPIKELET1 (IDS1)-like genes SUPERNUMERARY BRACT and OsIDS1.

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice (Oryza sativa L.)

A novel zebra mutant, zebra-15, derived from the restorer line Jinhui10 (Oryza sativa L. ssp. indica) treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased (55.4%) and the ratio of Chla/Chlb increased (90.2%) significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F(2) segregation populations derived from the cross of Xinong1A and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the Xinong1A and the zebra-15, their F(2) population was then used for gene mapping. Zebra-15 (Z-15) gene was primarily restricted on the short arm of chromosome 5 by 150 F(2) recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F(2) recessive individuals.

Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.

Genetic Analysis and Gene Mapping of a Novel Rolled-Leaf Mutant rl12(t) in Rice

Abstract Leaf is an important organ for photosynthesis. Moderate leaf rolling could facilitate structure improvement of plant population and enhance light-use efficiency, which is important in breeding for ideotype plants. A rolled leaf mutant temporarily named rl12(t) , was obtained from the rice ( Oryza sativa L.) restorer line Jinhui 10 treated with ethyl methyl sulphonate (EMS). In the mutant, the newly developing leaves of the mutant did not roll, the upper 1/3 section of mature leaves was curled, and the older mature leaves were rolled completely. The pigment contents of the mutant increased significantly. The cytoplasmic male sterile (CMS) line Xinong 1A with flat leaves was crossed with the rl12(t) mutant to produce F 1 and F 2 populations. Genetic analysis indicated that the mutant was controlled by a single dominant gene. Gene rl12(t) was finally located on chromosome 10 between SWU-1 and SWU-2 with the genetic distances of 1.5 and 0.2 cM, respectively. Because no genes for rolled leaf trait have been previously located on this chromosome, RL12(t) should be a novel and unique dominant gene for rolled leaf.

Gene mapping related to yellow green leaf in a mutant line in rice (Oryza sativa L.)

A mutant, which derived from the restorer line Jinhui10 treated with EMS, showed completely yellow green leaves, and it had low chlorophyll content and poor agronomic characteristics during the growing stage. The F1 plants from the cross between normal × the mutant showed normal green leaves, and the segregation ratio of normal to yellow green leaves was 3 : 1 in F2 population. It indicated that the trait was controlled by a single recessive nuclear gene, temporarily designated asygl3. The geneygl3 was mapped between RM468 and RM3684 with genetic distances 8.4 cM and 1.8 cM on chromosome 3. This result would be used as genetic information for fine mapping and map-based cloning ofygl3 gene.

Identification and Gene Mapping of a multi-floret spikelet 1 (mfs1) Mutant Associated with Spikelet Development in Rice

Abstract In this study, a rice spikelet mutant, multi-floret spikelet 1 ( mfs1 ), which was derived from ethylmethane sulfonate (EMS)-treated Jinhui 10 ( Oryza sativa L. ssp. indica) exhibited pleiotropic defects in spikelet development. The mfs1 spikelet displayed degenerated the empty glume, elongated the rachilla, the extra lemma-like organ and degraded the palea. Additionally, mfs1 flowers produced varied numbers of inner floral organs. The genetic analysis revealed that the mutational trait was controlled by a single recessive gene. With 401 recessive individuals from the F 2 segregation population, the MFS1 gene was finally mapped on chromosome 5, an approximate 350 kb region. The present study will be useful for cloning and functional analysis of MFS1 , which would facilitate understanding of the molecular mechanism involved in spikelet development in rice.

Physiological character and molecular mapping of leaf-color mutant wyv1 in rice (Oryza sativa L.)

The seed of an excellent indica restorer line Jinhui10 (Oryza sativa L. ssp. indica) was treated by ethyl methanesulfonate (EMS); a leaf-color mutant displaying distinct phenotype throughout development grown in paddy field was identified from the progeny. The mutant leaf showed white-yellow at seedling stage and then turned to yellow-green at tillering stage, after that, virescent color appeared until to maturity. The mutant was thus temporarily designed as wyv1. The chlorophyll contents decreased significantly and the changing was consistent with the chlorotic level of wyv1 leaves. Chlorophyll fluorescence kinetic parameters measured at the seedling stage showed that co-efficiency of photochemical quenching (qP), actual photosystem II efficiency (ΦPS II), electron transport rate (ETR) and initial chlorophyll fluorescence level (Fo), net photosynthetic rate (Pn) and maximum photochemical efficiency (Fv / Fm) significantly decreased in severe chlorotic leaf of the mutant compared with that of wild type. However, no significant differences were observed for Pn and Fv/Fm between virescent leaf and normal green leaf. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene which was finally mapped between SSR marker Y7 and Y6 on rice chromosome 3 based on F2 population of Xinong1A / wyv1. Genetic distances were 0.06 cM and 0.03 cM respectively, and the physical distance was 84 kb according to the sequence of indica rice 9311. The results must facilitate map-based cloning and functional analysis of WYV1 gene.

LATERAL FLORET 1 induced the three-florets spikelet in rice

Significance In cereal crops, the number of florets in a spikelet is an important factor affecting the grain number per panicle and then the grain yield. In wild-type rice, one spikelet produces one fertile floret. This study characterized a gain-of-function mutant lateral florets 1 (lf1) in rice. In lf1, the spikelet developed lateral florets with proper floral organ identities in the axil of the sterile lemma, showing that the rice spikelet has the potential to restore the “three-florets spikelet” which may have existed in ancestors. Therefore, it provides strong evidence supporting the three-florets spikelet hypothesis and presents a prospect for increasing grain number per panicle by breeding rice with three-floret spikelets. The spikelet is a unique inflorescence structure in grass. The molecular mechanisms behind the development and evolution of the spikelet are far from clear. In this study, a dominant rice mutant, lateral florets 1 (lf1), was characterized. In the lf1 spikelet, lateral floral meristems were promoted unexpectedly and could generally blossom into relatively normal florets. LF1 encoded a class III homeodomain-leucine zipper (HD-ZIP III) protein, and the site of mutation in lf1 was located in a putative miRNA165/166 target sequence. Ectopic expression of both LF1 and the meristem maintenance gene OSH1 was detected in the axil of the sterile lemma primordia of the lf1 spikelet. Furthermore, the promoter of OSH1 could be bound directly by LF1 protein. Collectively, these results indicate that the mutation of LF1 induces ectopic expression of OSH1, which results in the initiation of lateral meristems to generate lateral florets in the axil of the sterile lemma. This study thus offers strong evidence in support of the “three-florets spikelet” hypothesis in rice.